Custom pcr arrays

Total RNA from lung tissue was extracted using RNeasy-mini spin columns (Qiagen) after grinding in liquid nitrogen. RNA integrity and concentrations were estimated using RNA-nanochips on Bio-analyzer (Agilent). Extracted RNA was converted to cDNA by reverse transcriptase (RT² First Strand Kit, Qiagen). Quantitative PCR was performed using CFX connect (Bio-Rad) using custom PCR-arrays (Qiagen) for following genes of interest: IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12a, IL-13, IL-17a, IL-17f, IL-22, IL-23a, Ifnγ, Tnfα. Housekeeping genes included were: Ywhag, Actb, Polr2b, Gapdh, Sdha, and Tbp. Additionally, a random genomic DNA contamination control, three reverse transcriptase controls, and three additional PCR controls were used. Data was validated on independently extracted RNA and analyzed using in-house primers (primer sequences available on request) using SYBRGreen assay. Data was analyzed using comparative C_T method as described earlier (Kumar-Singh et al., 2006 (link); Schmittgen and Livak, 2008 (link)). Briefly, the combined average Ct value of the control group was used to calculate the fold differences in the study groups.

We used commercial 384-well Custom PCR arrays (Qiagen) which covered a total of 377 unique human miRNAs, 308 of which were conserved in cow (2 nucleotide mismatch allowed). Three pools of 3 to 4 samples each, from each of Day 0, Day 8 and Day 16 for non-pregnant animals, and from Day 24 pregnant animals (12 pools in total) were analysed. cDNA (10 μL) was synthesised from 2 μL RNA sample using miScript II RT kit (Qiagen) in a Whatman-Biometra Thermocycler (Biometra, USA). The arrays were setup according to the manufacturer’s instructions and were run on the LightCycler 480 System (Roche, Switzerland). Data analysis was performed using Microsoft Excel and R programming. Raw Cq data were initially filtered to remove wells with non-specific amplification as identified by melting-curve analysis. Next, miRNAs which had Cq > 35 in more than 75 % of samples per experimental group were removed from the dataset. Cq-values were normalised using the global mean expression, which was calculated from the miRNAs which were detected in all of the sample pools. The mean expression across non-pregnant time-points (Days 0, 8 and 16) was used for analyses, as for the sequencing data, resulting in 3 data-points for analyses from each of pregnant and non-pregnant groups (NP vs P24). The statistical analysis of the transformed normalised data was performed as described for the sequencing data above.